The architecture of the diaminobutyrate acetyltransferase active site provides 
mechanistic insight into the biosynthesis of the chemical chaperone ectoine

Richter, Alexandra A.; Kobus, Stefanie; Czech, Laura; Hoeppner, Astrid; Zarzycki, Jan; Erb, Tobias J.; Lauterbach, Lukas; Dickschat, Jeroen S.; Bremer, Erhard; Smits, Sander H. J.

doi:10.1074/jbc.RA119.011277

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The architecture of the diaminobutyrate acetyltransferase active site provides mechanistic insight into the biosynthesis of the chemical chaperone ectoine

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Zarzycki, Jan
Understanding and Building Metabolism, Department of Biochemistry and Synthetic Metabolism, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

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Erb, Tobias J.
Understanding and Building Metabolism, Department of Biochemistry and Synthetic Metabolism, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

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Citation

Richter, A. A., Kobus, S., Czech, L., Hoeppner, A., Zarzycki, J., Erb, T. J., et al. (2020). The architecture of the diaminobutyrate acetyltransferase active site provides mechanistic insight into the biosynthesis of the chemical chaperone ectoine. JOURNAL OF BIOLOGICAL CHEMISTRY, 295(9), 2822-2838. doi:10.1074/jbc.RA119.011277.

Cite as: https://hdl.handle.net/21.11116/0000-0008-BEA0-D

Abstract

Ectoine is a solute compatible with the physiologies of both prokaryotic
and eukaryotic cells and is widely synthesized by bacteria as an osmotic
stress protectant. Because it preserves functional attributes of
proteins and macromolecular complexes, it is considered a chemical
chaperone and has found numerous practical applications. However, the
mechanism of its biosynthesis is incompletely understood. The second
step in ectoine biosynthesis is catalyzed by l-2,4-diaminobutyrate
acetyltransferase (EctA; EC 2.3.1.178), which transfers the acetyl group
from acetyl-CoA to EctB-formed l-2,4-diaminobutyrate (DAB), yielding
N-?-acetyl-l-2,4-diaminobutyrate (N-?-ADABA), the substrate of ectoine
synthase (EctC). Here, we report the biochemical and structural
characterization of the EctA enzyme from the thermotolerant bacterium
Paenibacillus lautus (Pl). We found that (Pl)EctA forms a homodimer
whose enzyme activity is highly regiospecific by producing N-?-ADABA but
not the ectoine catabolic intermediate N-?-acetyl-l-2,4-diaminobutyric
acid. High-resolution crystal structures of (Pl)EctA (at 1.2?2.2 ?
resolution) (i) for its apo-form, (ii) in complex with CoA, (iii) in
complex with DAB, (iv) in complex with both CoA and DAB, and (v) in the
presence of the product N-?-ADABA were obtained. To pinpoint residues
involved in DAB binding, we probed the structure-function relationship
of (Pl)EctA by site-directed mutagenesis. Phylogenomics shows that
EctA-type proteins from both Bacteria and Archaea are evolutionarily
highly conserved, including catalytically important residues.
Collectively, our biochemical and structural findings yielded detailed
insights into the catalytic core of the EctA enzyme that laid the
foundation for unraveling its reaction mechanism.